Method and system for characterization and mapping of tissue lesions

ABSTRACT

The present invention provides a method and an apparatus for the in vivo, non-invasive, early detection of alterations and mapping of the grade of these alterations, caused in the biochemical and/or in the functional characteristics of epithelial tissues during the development of tissue atypias, dysplasias, neoplasias and cancers. The method is based, at least in part, on the simultaneous measurement of the spatial, temporal and spectral alterations in the characteristics of the light that is re-emitted from the tissue under examination, as a result of a combined tissue excitation with light and special chemical agents. The topical or systematic administration of these agents result in an evanescent contrast enhancement between normal and abnormal areas of tissue. The apparatus enables the capturing of temporally successive imaging in one or more spectral bands simultaneously. Based on the measured data, the characteristic curves that express the agent-tissue interaction kinetics, as well as numerical parameters derived from these data, are determined in any spatial point of the examined area. Mapping and characterization of the lesion, are based on these parameters.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional ApplicationNo. XXXXXX, entitled “Method And Apparatus For Amplifying PathologicalFeatures In Tissues”, filed on Dec. 15, 1999 and Greek NationalApplication Serial No. 20000100102, filed on Mar. 28, 2000, incorporatedherein in their entirety by this reference.

FIELD OF THE INVENTION

[0002] The present invention is directed to a method and apparatus forthe in vivo, non invasive detection and mapping of the biochemicaland/or functional pathologic alterations of human tissues.

BACKGROUND OF THE INVENTION

[0003] Cancer precursors signs are the so called pre-cancerous states,which are curable if they are detected at an early stage. In theopposite case the lesion can progress in depth, resulting in thedevelopment of invasive cancer and metastases. At this stage, thepossibilities of successful therapy are dramatically diminished.Consequently, the early detection and the objective identification ofthe severity (stage) of the precancerous lesion are of crucialimportance.

[0004] The conventional clinical process of optical examination havevery limited capabilities in detecting cancerous and pre-canceroustissue lesions. This is due to the fact that the structural andmetabolic changes, which take place during the development of thedecease, do not significantly and with specificity alter the colorcharacteristics of the pathological tissue.

[0005] In order to obtain more accurate diagnosis, biopsy samples areobtained from suspicious areas, which are submitted for histologicalexamination. However, biopsy sampling poses several problems, such as :a) risk for sampling errors associated with the visual limitations indetecting and localizing suspicious areas; b) biopsy can alter thenatural history of the intraepithelial lesion; c) mapping and monitoringof the lesion require multiple tissue sampling, which is subjected toseveral risks and limitations; d) the diagnostic procedure performedwith biopsy sampling and histologic evaluation is qualitative,subjective, time consuming, costly and labor intensive. In recent yearsthere have been developed and presented quite a few new methods andsystems in an effort to overcome the disadvantages of the conventionaldiagnostic procedures. These methods can be classified in twocategories: a) Methods which are based on the spectral analysis oftissues in vivo, in an attempt to improve the diagnostic information b)Methods which are based on the chemical excitation of tissues with theaid of special agents, which have the property to interact withpathologic tissue and to alter its optical characteristics selectively,thus enhancing the contrast between lesion and healthy tissue.

[0006] In the first case, the experimental use of spectroscopictechniques as a potential solutions to existing diagnostic problems, ismotivated by their capability to detect alterations in the biochemicaland/or the structural characteristics, which take place in the tissueduring the development of the disease. In particular, fluorescencespectroscopy has been extensively used in various tissues, where thelater are optically excited with the aid of a light source (usuallylaser), of short wave length (blue-ultraviolet range) and their responseis measured as fluorescence intensity vs. wavelength.

[0007] Garfield and Glassman in U.S. Pat. No. 5,450,857 and Ramanajum etal in U.S. Pat. No. 5,421,339 have presented a method based on the useof fluorescence spectroscopy for the diagnosis of cancerous andpre-cancerous lesions of cervix. The main disadvantage of fluorescencespectroscopy is that the existing biochemical modifications associatedwith the progress of the disease are not manifested in a direct way asmodifications in the measured fluorescence spectra. The fluorescencespectra contain limited diagnostic information of two basic reasons: a)Tissues contain non-fluorescent chromophores, such as hemoglobin.Absorption by such chromophores of the emitted light from fluorophorescan result in artificial dips and peaks in the fluorescence spectra. Inother words the spectra carry convoluted information for severalcomponents and therefore it is difficult assess alterations in tissuefeatures of diagnostic importance; and b) The spectra are broad due tothe fact that a large number of tissue components are optically excitedand contribute to the captured optical signal. As a result the spectrado not carry specific information for the pathologic alterations andthus they are of limited diagnostic value.

[0008] The latter is expressed as low sensitivity and specificity in thedetection and classification of tissue lesions.

[0009] Aiming to enhance the sensitivity and specificity of the capturedinformation, Ramanujan et al in the Patent No. WO 98/24369 havepresented a method based on the use of neural networks for the analysisof the spectral data. This method is based on the training of acomputing system with a large number of spectral patterns, which havebeen taken from normal and from pathologic tissues. The spectrum that iscaptured each time is compared with the stored spectral data,facilitating this way, the identification of the tissue pathology.

[0010] R. R. Kortun et al, in U.S. Pat. No. 5,697,373, seeking toimprove the captured diagnostic information, have presented a methodbased on the combination of fluorescence spectroscopy and Ramanscattering. The last has the capability of providing more analyticalinformation, it requires however complex instrumentation and idealexperimental conditions, which substantially hinder their clinical use.It is generally known that tissues are characterized by the lack ofspatial homogeneity and consequently the spectral analysis ofdistributed spatial points is insufficient for the characterization oftheir status.

[0011] Dombrowski in U.S. Pat. No. 5,424,543, describes amulti-wavelength, imaging system, capable of capturing tissue images inseveral spectral bands. With the aid of such a system it is possible ingeneral to map characteristics of diagnostic importance based on theirparticular spectral characteristics. However, due to the insignificanceof the spectral differences between normal and pathologic tissue, whichis in general the case, inspection in narrow spectral bands does notallow the highlighting of these characteristics and even more so, theidentification and staging of the pathologic area.

[0012] D. R. Sandison et al, in U.S. Pat. No. 5,920,399 describe animaging system, developed for the in vivo investigation of cells, whichcombines multi-band imaging and light excitation of the tissue. Thesystem also employs a dual fiber optic bundle for the transferring ofthe emitted from the source light onto the tissue and the remitted lightfrom the tissue to the optical detector. These bundles are placed incontact with the tissue, and various wavelengths of excitation andimaging are combined in attempt to enhance the spectral differentiationbetween normal and pathologic tissue.

[0013] In U.S. Pat. No. 5,921,926, J. R. Delfyett et al have presented amethod for the diagnosis of diseases of the cervix, which is based onthe combination of Spectral Interferometry and Optical CoherenceTomography (OCT). This system combines three-dimensional imaging andspectral analysis of the tissue.

[0014] Moreover, several improved versions of colposcopes have beenpresented, (D. R.Craine et al, U.S. Pat. No. 5,791,346 and K. L. BlaizU.S. Pat. No. 5,989,184) in most of which, electronic imaging systemshave been integrated for image capturing, analysis of tissue images,including the quantitative assessment of lesion's size. For theenhancement of the optical differentiation between normal and pathologictissue, special agents are used in various fields of biomedicaldiagnostics, which are administered topically or systematically. Suchagents are acetic acid solution, toluidine blue, variousphotosensitizers (porphyrines) (S. Anderson Engels, C. Klinteberg, K.Svanberg, S. Svanberg, In vivo fluorescence imaging for tissuediagnostics, Phys Med. Biol. 42 (1997) 815-24). The provoked selectivestaining of the pathologic tissue is owed to the property of theseagents to interact with the altered metabolic and structuralcharacteristics of the pathologic area. This interaction enhancesprogressively and reversibly the differences in the spectralcharacteristics of reflection and/or fluorescence between normal andpathologic tissue. Despite the fact that the provoked selective stainingof the pathologic tissue is a dynamic phenomenon, in clinical practicethe intensity and the extent of the staining are assessed qualitativelyand statically. Furthermore, in several cases of early pathologicconditions, the phenomenon of temporary staining after administering theagent, is short-lasting and thus the examiner is not able to detect theprovoked alterations and even more so, to assess their intensity andextent. In other cases, the staining of the tissue progresses veryslowly, with the consequence of patient discomfort and creation ofproblems for the examiner in assessing the intensity and extent of thealterations, since they are continuously changing. The above have asdirect consequence, the downgrading of the diagnostic value of thesediagnostic procedures and thus its usefulness is limited to facilitatethe localization of suspected areas for obtaining biopsy samples.

[0015] Summarizing the above mentioned, the following conclusions aredrawn:

[0016] a) Various conventional light dispersion spectroscopic techniques(fluorescence, elastic, non elastic scattering, etc) have been proposedand experimentally used for the in vivo detection of alterations in thestructural characteristics of pathologic tissue. The main disadvantageof these techniques is that they provide point information, which isinadequate for the analysis of the spatially non-homogenous tissue.Multi-band imaging has the potential to solve this problem, by providingspectral information (of lesser resolution as a rule) but in any spatialpoint of the area under examination. These techniques (imaging andnon-imaging) however, provide information of limited diagnostic value,due to the fact that the structural tissue alterations, which areaccompanying the development of the disease, are not manifested assignificant and characteristic alterations on the measured spectra.Consequently, the captured spectral information cannot be directlycorrelated with the tissue pathology, a fact which limits the clinicalusefulness of these techniques.

[0017] b) The conventional (non-spectral) imaging techniques provide thecapability of mapping characteristics of diagnostic importance in two orthree dimensions. They are basically used for measuring morphologicalcharacteristics and as clinical documentation tools.

[0018] c) The diagnostic methods which are based on the selectivestaining of pathologic tissue with special agents allows the enhancementof the optical contrast between normal and pathologic tissue.Nevertheless they provide limited information for the in vivoidentification and staging of the disease.

[0019] Given the fact that the selective interaction of pathologictissue with the agents, which enhance its optical contrast with healthytissue is a dynamic phenomenon, it is reasonable to suggest that thecapture and analysis of the characteristics of this phenomenon'skinetics, could provide important information for the in vivo detection,identification and staging of tissue lesions. In a previous publicationby the inventors (C. Balas, A. Dimoka, E. Orfanoudaki, E. koumandakis,“In vivo assessment of acetic acid-cervical tissue interaction usingquantitative imaging of back-scattered light: Its potential use for thein vivo cervical cancer detection grading and mapping”, SPIE-OpticalBiopsies and Microscopic Techniques, Vol. 3568 pp. 31-37, (1998)),measurements of the alterations in the characteristics of theback-scattered light as a function of wave-length and time arepresented. These alterations are provoked in the cervix by the topicaladministration of acetic acid solution. In this particular case, therewas used as an experimental apparatus, a general-purpose multi-spectralimaging system built around a tunable liquid crystal monochromator formeasuring the variations in intensity of the back-scattered light as afunction of time and wavelength in selected spatial points. It was foundthat the lineshapes of curves of intensity of back-scattered lightversus time, provide advanced information for the direct identificationand staging of tissue neoplasias. Unpublished results of the sameresearch team support that similar results can also be obtained withother agents, which have the property of enhancing the optical contrastbetween normal and pathologic tissue. Nevertheless, the experimentalmethod employed in the published paper is characterized by quite a fewdisadvantages, such as: The imaging monochromator requires time forchanging the imaging wavelength and as a consequence it is inappropriatefor multispectral imaging and analysis of dynamic phenomena. It does notconstitute a method for the mapping of the grade of the tissue lesions,as the presented curves illustrate the temporal alterations of intensityof the back-scattered light in selected points. The lack of datamodeling and parametric analysis of the characteristics of thephenomenon kinetics in any spatial point of the area of interestrestrict the usefulness of the method in experimental studies and hinderits clinical implementation. The optics used for the imaging of the areaof interest are of general purpose and are not comply with the specialtechnical requirements for the clinical implementation of the method.Clinical implementation of the presented system is also hindered by thefact that it does not integrate appropriate means for ensuring thestability of the relative position between the tissue surface and imagecapturing module, during the snapshot imaging procedure. This is veryimportant since small movements of the patient (i.e. breathing) arealways present during the examination procedure. If micro-movements aretaking place during successive capturing of images, after application ofthe agent, then the spatial features of the captured images are notcoincide. This reduces substantially the precision in the calculation ofthe curves in any spatial point, that express the kinetics ofmarker-tissue interaction.

SUMMARY OF THE INVENTION

[0020] The present invention provides, at least in part, a method formonitoring the effects of a pathology differentiating agent on a tissuesample by applying a pathology differentiating agent, e.g., acetic acid,on a tissue sample and monitoring the rate of change of light reflectionfrom the tissue sample over time, thereby monitoring the effects of apathology differentiating agent on a tissue sample. The tissue may be acervical, ear, oral, skin, esophagus, or stomach tissue. Withoutintending to be limited by theory, it is believed that the pathologydifferentiating agent provokes transient alterations in the lightscattering properties of the tissue, e.g., the abnormal epithelium.

[0021] In another aspect, the present invention features a method forthe in vivo diagnosis of a tissue abnormality, e.g., a tissue atypia, atissue dysplasia, a tissue neoplasia (such as a cervical intraepithelialneoplasia, CINI, CINII, CINIII) or cancer, in a subject. The methodincludes contacting a tissue in a subject with a pathologydifferentiating agent, e.g., an acetic acid solution or a combination ofsolutions selected from a plurality of acidic and basic solutions,exposing the tissue in the subject to optical radiation; and monitoringthe intensity of light emitted from the tissue over time, therebydiagnosing a tissue abnormality in a subject. The optical radiation maybe broad band optical radiation, preferably polarized optical radiation.

[0022] The non-invasive methods of the present invention are useful forthe in vivo early detection of tissue abnormalities/alterations andmapping of the grade of these tissue abnormalities/alterations, causedin the biochemical and/or in the functional characteristics ofepithelial tissues, during the development of tissue atypias,dysplasias, neoplasias and cancers.

[0023] In one embodiment, the tissue area of interest is illuminatedwith a broad band optical radiation and contacted with a pathologydifferentiating agent, e.g., an agent or a combination of agents whichinteract with pathologic tissue areas characterized by an alteredbiochemical composition and/or cellular functionality and provoke atransient alteration in the characteristics of the light that isre-emitted from the tissue. The light that is re-emitted from the tissuemay be in the form of reflection, diffuse scattering, fluorescence orcombinations or subcombinations thereof. The intensity of the lightemitted from the tissue may be measured, e.g., simultaneously, in everyspatial point of the tissue area of interest, at a given time point orover time (e.g., for the duration of agent-tissue interaction). Adiagnosis may be made based on the quantitative assessment of thespatial distribution of alterations in the characteristics of the lightre-emitted from the tissue at given time points, before and after theoptical and chemical excitation of the tissue and/or based on thequantitative assessment of the spatial distribution of parameters,calculated from the characteristic curves that express the kinetics ofthe provoked alterations in the characteristics of the light re-emittedfrom the tissue, which characteristic curves are simultaneously measuredin every spatial point of the area under examination during the opticaland chemical excitation of the tissue.

[0024] In one embodiment of the invention, the step of tissueillumination comprises exposing the tissue area under analysis tooptical radiation of narrower spectral width than the spectral width ofthe light emitted by the illumination source. In another embodiment, thestep of measuring the intensity of light comprises measuring theintensity of the re-emitted light in a spectral band, the spectral widthof which is narrower than the spectral width of the detector'ssensitivity. In yet another embodiment, the step of measuring theintensity of light comprises measuring simultaneously the intensity ofthe re-emitted light in a plurality of spectral bands, the spectralwidths of which are narrower than the spectral width of the detector'ssensitivity.

[0025] In yet another aspect, the present invention features anapparatus for the in vivo, non-invasive early detection of tissueabnormalities/alterations and mapping of the grade of these tissueabnormalities/alterations, caused in the biochemical and/or in thefunctional characteristics of epithelial tissues, during the developmentof tissue atypias, dysplasias, neoplasias and cancers. The apparatusincludes optics for collecting the light re-emitted by the area underanalysis, selecting magnification and focusing the image of the area;optical imaging detector(s); means for the modulation, transfer, displayand capturing of the image of the tissue area of interest; a computerwhich includes data storage, processing and analysis means; a monitorfor displaying images, curves and numerical data; optics for the opticalmultiplication of the image of the tissue area of interest; a lightsource for illuminating the area of interest; optionally, opticalfilters for selecting the spectral band of imaging and illumination;means for transmitting light and illuminating the area of interest;control electronics; and optionally, software for the analysis andprocessing of data, which also enables the tissue image capturing andstoring in specific time points and for a plurality of time points,before and after administration of the pathology differentiating agent.

[0026] Using the foregoing apparatus an image or a series of images maybe created which express the spatial distribution of the characteristicsof the kinetics of the provoked changes in the tissue's opticalcharacteristics, before and after the administration of the agent, withpixel values corresponding with the spatial distribution of thealterations in the intensity of the light emitted from the tissue, ingiven time instances, before and after the optical and chemicalexcitation of tissue and/or with the spatial distribution of parametersderived from the function: pixel gray value versus time. The foregoingfunction may be calculated from the captured and stored images and foreach row of pixels with the same spatial coordinates.

[0027] In one embodiment, the step of optical filtering the imagingdetector comprises an optical filter that is placed in the optical pathof the rays that form the image of the tissue, for the recording oftemporally successive images in a selected spectral band, the spectralwidth of which is narrower than the spectral width of the detector'ssensitivity.

[0028] In yet another embodiment, the image multiplication opticscomprise light beam splitting optics that create two identical images ofthe area of interest, which are recorded by two imaging detectors, infront of which optical filters are placed, with in general differenttransmission characteristics and capable of transmitting light ofspectral width shorter than the spectral width of the detector'ssensitivity, so that two groups of temporally successive images of thesame tissue area are recorded simultaneously, each one corresponding toa different spectral band.

[0029] In another embodiment, the image multiplication optics comprisemore than one beam splitter for the creation of multiple identicalimages of the area of interest, which are recorded by multiple imagingdetectors, in front of which optical filters are placed, with,preferably, different transmission characteristics and capable oftransmitting light of spectral width shorter than the spectral width ofthe detector's sensitivity, so that multiple groups of temporallysuccessive images of the same tissue area are recorded simultaneously,each one corresponding to a different spectral band.

[0030] In a further embodiment, the image multiplication optics compriseone beam splitter for the creation of multiple identical images of thearea of interest, which are recorded by multiple imaging detectors, infront of which optical filters are placed with, preferably, differenttransmission characteristics and capable of transmitting light ofspectral width shorter than the spectral width of the detector'ssensitivity, so that multiple groups of temporally successive images ofthe same tissue area are recorded simultaneously, each one correspondingto a different spectral band.

[0031] In yet a further embodiment, the image multiplication opticscomprise one beam splitter for the creation of multiple identical imagesof the area of interest, which are recorded in different sub-areas ofthe same detector, and in front these areas optical filters are placedwith, preferably, different transmission characteristics and capable oftransmitting light of spectral width shorter than the spectral width ofthe detector's sensitivity, so that multiple groups of temporallysuccessive images of the same tissue area are recorded simultaneously inthe different areas of the detector, each one corresponding to adifferent spectral band.

[0032] In another embodiment, the step of filtering the light sourcecomprises an optical filter, which is placed in the optical path of anillumination light beam, and transmits light of spectral width shorterthan the spectral width of sensitivity of the detector used.

[0033] In a further embodiment, the step of filtering the light sourcecomprises a plurality of optical filters and a mechanism for selectingthe filter that is interposed to the tissue illumination optical path,thus enabling the tuning of the center wavelength and the spectral widthof the light illuminating the tissue.

[0034] In another embodiment, the mapping of the grade of thealterations to the biochemical and/or functional characteristics of thetissue area of interest, is based on the pixel values of one image, fromthe group of the recorded temporally successive images of the tissuearea of interest.

[0035] In a further embodiment, the mapping of the grade of thealterations to the biochemical and/or functional characteristics of thetissue area of interest, is based on the pixel values belonging toplurality of images, which are members of the group of the recordedtemporally successive images of the tissue area of interest.

[0036] In another embodiment, the mapping of the grade of thealterations to the biochemical and/or functional characteristics of thetissue area of interest, is based on numerical data derived frommathematical operations and calculations between the pixel valuesbelonging a plurality of images, which are members of the group of therecorded temporally successive images of the tissue area of interest.

[0037] In a further embodiment, a pseudo-color scale, which representswith different colors the different pixel values of the image or of theimages used for the mapping of abnormal tissue areas, is used for thevisualization of the mapping of the grade of the alterations to thebiochemical and /or functional characteristics of the tissue area underexamination.

[0038] In one embodiment, the image or images which are determined forthe mapping of the grade of the alterations in biochemical and/orfunctional characteristics of tissue, are used for the in vivodetection, mapping, as well as for the determination of the borders ofepithelial lesions.

[0039] In another embodiment, the pixel values of the image or of theimages which are determined for the mapping of the grade of alterationsin biochemical and/or functional characteristics of tissue, are used asdiagnostic indices for the in vivo identification and staging ofepithelial lesions.

[0040] In yet another embodiment, the image or the images which aredetermined for the mapping of the grade of the alterations inbiochemical and/or functional characteristics of tissue can beoverimposed onto the color or black and white image of the same area oftissue under examination displayed on the monitor, so that abnormaltissue areas are highlighted and their borders are demarcated,facilitating the selection of a representative area for taking a biopsysample, the selective surgical removal of the abnormal area and theevaluation of the accuracy in selecting and removing the appropriatesection of the tissue.

[0041] In a further embodiment, the image or the images which aredetermined for the mapping of the grade of alterations in biochemicaland/or functional characteristics of tissue are used for the evaluationof the effectiveness of various therapeutic modalities such asradiotherapy, nuclear medicine treatments, pharmacological therapy, andchemotherapy.

[0042] In another embodiment, the optics for collecting the lightre-emitted by the area under analysis, comprises the optomechanicalcomponents employed in microscopes used in clinical diagnosticexaminations, surgical microscopes, colposcopes and endoscopes.

[0043] In one embodiment of the invention, for colposcopy applications,the apparatus may comprise a speculum, an articulated arm onto which theoptical head is attached, which optical head comprises a refractiveobjective lens, focusing optics, a mechanism for selecting themagnification, an eyepiece, a mount for attaching a camera, and anilluminator, where the speculum is attached in a fixed location onto thesystem articulated arm-optical head, in such a way such that the centrallongitudinal axis of the speculum is perpendicular to the central areaof the objective lens, so that when the speculum is inserted into thevagina and fixed in it, the relative position of the image-capturingoptics and of the tissue area of interest remains unaltered, regardlessof micro-movements of the cervix, which are taking place during theexamination of the female subject.

[0044] In a further embodiment, the apparatus may further comprise anatomizer for delivering the agent, where the atomizer is attached in afixed point onto the system articulated arm-optical head of theapparatus and in front of the vaginal opening, where the spraying of thetissue may be controlled and synchronized with a temporally successiveimage capturing procedure, with the aid of electronic control means.

[0045] In another embodiment of the apparatus of the invention, theimage capturing detector means and image display means comprise a camerasystem with detector spatial resolution greater than 1000×1000 pixelsand a monitor of at least 17 inches (diagonal), so that highmagnification is ensured together with a large field of view, while theimage quality is maintained.

[0046] In a further embodiment, in the case of microscopes used inclinical diagnostic examinations, surgical microscopes and colposcopes,comprise an articulated arm onto which the optical head is attached,which optical head comprises an objective lens, focusing optics, amechanism for selecting the magnification, an eyepiece, a mount forattaching a camera, an illuminator and two linear polarizers, where thetwo linear polarizers are attached, one at a point along the opticalpath of the illuminating light beam and the other at a point along theoptical path of the rays that form the image of the tissue, with thecapability of rotating the polarization planes of these light polarizingoptical elements, so that when these planes are perpendicular to eachother, the contribution of the tissue's surface reflection to the formedimage is eliminated.

[0047] In another embodiment, in the case of endoscopy, the endoscopemay comprise optical means for transferring light from the light sourceonto the tissue surface and for collecting and transferring along almostthe same axis and focusing the rays that form the image of the tissue,and two linear polarizers, where the two linear polarizers are attached,one at a point along the optical path of the illuminating light beam andthe other at a point along the optical path of the rays that form theimage of the tissue, with the capability of rotating the polarizationplanes of these light polarizing optical elements, so that when theseplanes are perpendicular to each other, the contribution of the tissue'ssurface reflection to the formed by the endoscope image is eliminated.

[0048] In another embodiment, in the case of microscopes used inclinical diagnostic examinations, surgical microscopes and colposcopes,may additionally comprise a reflective objective lens, where thereflective objective replaces the refractive one, which reflectiveobjective is devised so that in the central part of its optical frontaperture the second reflection mirror is located, and in the rear part(non-reflective) of this mirror, illumination means are attached fromwhich light is emitted toward the object, so that with or withoutillumination beam zooming and focusing optics the central ray of theemitted light cone is coaxial, with the central ray of the light beamthat enters the imaging lens, and with the aid of zooming and focusingoptics of illumination beam that may be adjusted simultaneously andautomatically with the mechanism for varying the magnification of theoptical imaging system, the illuminated area and the field-of-view ofthe imaging system, are varying simultaneously and proportionally, sothat any decrease in image brightness caused by increasing themagnification, is compensated with the simultaneous zooming and focusingof the illumination beam.

[0049] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1 is a schematic representation of the present method's basicprinciple.

[0051]FIG. 2, illustrates an embodiment of the invention comprising amethod for capturing in two spectral bands simultaneously and in anyspatial point of the area under analysis, the kinetics of thealterations in the characteristics of the remitted from the tissuelight, before and the after the administration of the contrast enhancingagent

[0052]FIG. 3 illustrates another embodiment of the invention comprisinga method for capturing in different spectral bands simultaneously and inany spatial point of the area under analysis, the kinetics of thealterations in the characteristics of the remitted from the tissuelight, before and the after the administration of the contrast enhancingagent.

[0053]FIG. 4 illustrates a schematic diagram of a medical microscopecomprising a light source (LS), a magnification selection mechanism(MS), an eyepiece (EP) and a mount for attaching the image capturingmodule (CA), (detector(s), readout electronics etc).

[0054]FIG. 5 illustrates an endoscope comprising an eyepiece (EP), whichcan be adapted to an electronic imaging system, optical fibers orcrystals for the transmission of both illumination and image rays,optics for the linear polarization of light, one interposed to theoptical path of the illumination rays (LE) and one to the path of theray that form the optical image of the tissue (II).

[0055]FIG. 6 depicts a colposcopic apparatus comprising an articulatedarm (AA), onto which the optical head (OH) is affixed, which includes alight source (LS), an objective lens (OBJ), an eye-piece (EP) and opticsfor selecting the magnification (MS).

[0056]FIG. 7 illustrates an optical imaging apparatus which comprises alight source located at the central part of its front-aperture.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention is directed to a method and system for thein-vivo, non-invasive detection and mapping of the biochemical and orfunctional alterations of tissue, e.g., tissue within a subject. Uponselection of the appropriate agent which enhances the optical contrastbetween normal and pathologic tissue (depending on the tissue'spathology), this agent is administered, e.g., topically to the tissue.In FIG. 1, the tissue (T), is sprayed using an atomizer (A), whichcontains the agent, e.g., acetic acid. At the same time, the tissue isilluminated with a source that emits light at a specific spectral band,depending on the optical characteristics of both the agent and thetissue. Illumination and selection of the spectral characteristics ofthe incident to the tissue light can be performed with the aid of alight source (LS) and a mechanism for selecting optical filters (OFS).Of course there are several other methods for illuminating the tissueand for selecting the spectral characteristics of the incident light(e.g., Light emission diodes, LASERS and the like).

[0058] For the imaging of the area of interest, light collection optics(L) are used, which focus the image onto a two-dimensional opticaldetector (D). The output signal of the latter is amplified, modulatedand digitized with the aid of appropriate electronics (EIS) and finallythe image is displayed on a monitor (M) and stored in the data-storingmeans of a personal computer (PC). Between tissue (T) and detector (D),optical filters (OFI) can be interposed. The interposition of the filtercan be performed for tissue (T) imaging in selected spectral bands, atwhich the maximum contrast is obtained between areas that are subjectedto different grade of alterations in their optical characteristics,provoked after administering the appropriate agent.

[0059] Before administration of the latter, images can be captured andused as reference. After the agent has been administered, the detector(D), captures images of the tissue, in successive time instances, whichare then stored in the computer's data-storage means. The capturing rateis proportional to the rate at which the tissue's opticalcharacteristics are altered, following the administration of the agent.

[0060] In FIG. 1, images of the same tissue area are schematicallyillustrated, which have been stored successively before and afteradministering the agent (STI). In these images, the black areasrepresent tissue areas that do not alter their optical characteristics(NAT), while the gray-white tones represent areas which alter theiroptical characteristics (AT), following the administration of the agent.The simultaneous capture of the intensity of the light re-emitted fromevery spatial point of the tissue area under analysis and inpredetermined time instances, allows the calculation of the kinetics ofthe provoked alterations.

[0061] In FIG. 1, two curves are illustrated: pixel value in position xy(Pvxy), versus time t. The curve ATC corresponds to an area where agentadministration provoked alterations (AT) in the tissue's opticalcharacteristics. The curve (NATC) corresponds to an area where noalteration took place (NAT).

[0062] The mathematical analysis of these curves, leads to thecalculation of quantitative parameters for every pixel such as: Thevalue PVxy that corresponds to the time ti, the relaxation time trelwhich corresponds to the value Pvxy=A/e (where e is the base of Neperlogarithms), etc.

[0063] The calculation of these parameters (P) in every spatial point ofthe area under analysis, allows the calculation of the image or imagesof the kinetics of the phenomenon (KI), with pixel values that arecorrelated with these parameters. These values can be represented with ascale of pseudocolors (Pmin, Pmax), the spatial distribution of whichallows for immediate optical evaluation of the intensity and extent ofthe provoked alterations. Depending on the correlation degree betweenthe intensity and the extent of the provoked alterations with thepathology and the stage of the tissue lesion, the measured quantitativedata and the derived parameters would allow the mapping, thecharacterization and the border-lining of the lesion. The pseudocolorimage of the phenomenon's kinetics (KI), which expresses the spatialdistribution of one or more parameters, can be overimposed (after beingcalculated) on the tissue image, which is displayed in real-time on themonitor. The using the overimposed image as a guide, facilitatessubstantially the determination of the lesion's boundaries, forsuccessful surgical removal of the entire lesion, or for locatingsuspicious areas in order to obtain a biopsy sample(s). Furthermore,based on the correlation of the phenomenon's kinetics with the pathologyof the tissue, the measured quantitative data and the parameters thatderive from them, can constitute quantitative clinical indices for thein vivo staging of the lesion or of sub-areas of the latter.

[0064] In some cases it is necessary to capture the kinetics of thephenomenon in more than one spectral band. This can serve in the in vivodetermination of illumination and/or imaging spectral bands at which themaximum diagnostic signal is obtained. Furthermore, the simultaneousimaging in more than one spectral bands can assist in minimizing thecontribution of the unwanted endogenous scattering, fluorescence andreflection of the tissue, to the optical signal captured by thedetector. The captured optical signal comprise the optical signalgenerated by the marker-tissue interaction and the light emitted fromthe endogenous components of the tissue. In many cases the recordedresponse of the components of the tissue constitute noise, since itoccludes the generated optical signal, which caries the diagnosticinformation. Therefore, separation of these signals, based on theirparticular spectral characteristics, will result in the maximization ofthe signal-to-noise ratio and consequently in the improvement of theobtained diagnostic information.

[0065]FIG. 2, illustrates a method for capturing in two spectral bandssimultaneously and in any spatial point of the area under analysis, thekinetics of the alterations in the characteristics of the remitted fromthe tissue light, before and the after the administration of thecontrast enhancing agent. The remitted from the tissue light, iscollected and focused by the optical imaging module (L) and passesthrough a beam splitting (BSP) optical element. Thus, two identicalimages of the tissue (T) are generated, which can be captured by twodetectors (D1, D2). In front of the detector, appropriate opticalfilters (Ofλ1), (Ofλ2) can be placed, so that images with differentspectral characteristics are captured. Besides beam splitters, opticalfilters, dichroic mirrors etc, can also be used for splitting the imageof the object. The detectors (D1), (D2) are synchronized so that theycapture simultaneously the corresponding spectral images of the tissue(Tiλ1), (Tiλ2) and in successive time-intervals, which are stored in thecomputer's data storage means. Generalizing, multiple spectral imagescan be captured simultaneously by combining multiple splitting elements,filters and sources.

[0066]FIG. 3 illustrates another method for capturing in differentspectral bands simultaneously and in any spatial point of the area underanalysis, the kinetics of the alterations in the characteristics of theremitted from the tissue light, before and the after the administrationof the contrast enhancing agent. With the aid of a special prism (MIP)and imaging optics, it is possible to form multiple copies of the sameimage onto the surface of the same detector (D). Various optical filters(OFλ1),(OFλ2),(OFλ3),(OFλ4), can be interposed along the length of theoptical path of the rays that form the copies of the object's image, sothat the captured multiple images correspond to different spectralareas.

[0067] For the clinical use of the methods of the invention, thedifferent implementations of image capturing module described above canbe integrated to conventional optical imaging diagnostic devises. Suchdevises are the various medical microscopes, colposcopes and endoscopes,which are routinely used for the in vivo diagnostic inspection oftissues. Imaging of internal tissues of the human body requires in mostcases the illumination and imaging rays to travel along the same opticalpath, through the cavities of the body. Due to this fact, in the commonoptical diagnostic devises the tissue's surface reflection contributessubstantially in the formed image. This limits the imaging informationfor the subsurface characteristics, which are in general of greatdiagnostic importance. This problem becomes more serious especially inepithelial tissues such as the cervix, larynx, oral cavity etc, whichare covered by fluids such as mucus and saliva. Surface reflection alsoobstructs the detection and the measurement of the alterations in thetissue's optical properties, provoked after the administration of agentswhich enhance the optical contrast between normal and pathologic tissue.More specifically, when a special agent alters selectively thescattering characteristics of the pathologic tissue, the strong surfacereflection that takes place in both pathologic (agent responsive) andnormal (agent non responsive) tissue areas, occludes the diagnosticsignal that originates from the interaction of the agent with thesubsurface features of the tissue. In other words, surface reflectionconstitutes optical noise in the diagnostic signal degradingsubstantially the perceived contrast between agent responsive and agentnon responsive tissue areas.

[0068] Based on the above, the effective integration of the method toimaging diagnostic devises, requires embodiments of appropriate opticsthat ensure the elimination of the contribution of surface reflection tothe captured image. FIG. 4 illustrates a schematic diagram of a medicalmicroscope consisted from a light source (LS), a magnification selectionmechanism (MS), an eyepiece (EP) and a mount for attaching the imagecapturing module (CA), (detector(s), readout electronics etc). For theelimination of the surface reflection a pair of linear polarizers isemployed. The incident to the tissue light (LS), is linearly polarizedby passing though a linear polarizer (LPO). The surface reflected light(TS), has the same polarization plane with the incident to the tissuelight (Fresnel reflection). By interposing the other linear polarizer tothe optical path of the rays that are remitted from the tissue and formthe optical image of the object, with its polarization planeperpendicular to the polarization level of the incident to the tissuelight (IPO), the contribution of the surface reflection to the image ofthe object is eliminated. The light which is not surface-reflectedenters the tissue, where due to multiple scattering, light polarizationis randomized. Thus, a portion of the re-emitted light passes throughthe imaging polarization optics, carrying improved information for thesubsurface features.

[0069]FIG. 5 illustrates an endoscope consisted of an eyepiece (EP),which can be adapted to an electronic imaging system, optical fibers orcrystals for the transmission of both illumination and image rays,optics for the linear polarization of light, one interposed to theoptical path of the illumination rays (LE) and one to the path of theray that form the optical image of the tissue (II). The polarizationplane of the polarizing optics, which are adapted to the exit of lightfrom the endoscope (LPO), is perpendicular to the polarization plane ofthe polarizer, which is adapted to the point where the light enters theendoscope (IL). The polarization optics of the incident to the tissuelight could also be adapted at the point where the light enters theendoscope (IL) but in this case, the endoscope has to be constructedusing polarization preserving crystals or fiber optics for transferringthe light. If polarization preserving light transmission media are used,then the polarizing optics of the imaging rays can be interposed intheir path and before or after the eyepiece (EP).

[0070] A problem for the effective clinical implementation of thedescribed method herein is the micro-movements of the patient, which arealways present during the snapshot imaging of the same tissue area.Obviously this problem is eliminated in case that the patient is underanesthesia (open surgery). In most cases however the movements of thetissue relative to the image capturing module, occurring during thesuccessive image capturing time-course, have the consequence that theimage pixels, with the same image coordinates, do not correspond toexactly the same spatial point x,y of the tissue area under examination.

[0071] This problem is typically encountered in colposcopy. A method toeliminate the influence to the measured temporal data of the relativemovements between tissue and image capturing module is presented below.A colposcopic apparatus is illustrated in FIG. 6, consisted of anarticulated arm (AA), onto which the optical head (OH) is affixed, whichincludes a light source (LS), an objective lens (OBJ), an eye-piece (EP)and optics for selecting the magnification (MS). The image capturingmodule is attached to the optical head (OH), through an opto-mechanicaladapter. A speculum (KD), which is used to open-up the vaginal canal forthe visualization of the cervix, is connected mechanically with theoptical head (OH), so that the its longitudinal symmetry axis (LA), tobe perpendicular to the central area of the objective lens (OBJ). Thespeculum enters the vagina and its blades are opened up compressing theside walls of the vagina. The Speculum (KD), been mechanically connectedwith the optical head (OH), transfer any micromovement of the patient tothe optical head (OH), which been mounted on an articulated arm (AA),follows these movements. Thus the relative position between tissue andoptical head remains almost constant.

[0072] An important issue that must also be addressed for the successfulclinical implementation of the diagnostic method described herein, isthe synchronization of the application of the contrast enhancing agentwith the initiation of the snapshot imaging procedure. FIG. 6,illustrates an atomizer (A) attached to the optical head of themicroscope. The unit (MIC) is comprised of electronics for controllingthe agent sprayer and it can incorporate also the container for storingthe agent. When the unit (MIC) receives the proper command from thecomputer it sprays a predetermined amount of the agent onto the tissuesurface, while the same or another command initiates the snapshot imagecapturing procedure.

[0073] The diagnostic examination of non-directly accessible tissues,located in cavities of the human body (ear, cervix, oral cavity,esophagus, colon, stomach), is performed with the aid of common clinicalmicroscopes. In these devises the illumination-imaging rays are nearco-axial. More specifically, the line perpendicular to the exit point oflight into the air, and the line perpendicular to the objective lens,form an angle of a few degrees. Due to this fact, these microscopesoperate at a specific distance from the subject (working distance), inwhich the illuminated tissue area, coincides with the field-of-view ofthe imaging system. These microscopes are found to be inappropriate incases where tissue imaging through human body cavities of small diameterand at short working distances, is required. These technical limitationsare also constituting serious restricting factors for the successfulclinical implementation of the method described herein. As it has beendiscussed above, elimination of surface reflection results in asubstantial improvement of the diagnostic information, obtained from thequantitative assessment of marker-tissue interaction kinetics. If acommon clinical microscope is employed as the optical imaging module,then due the above mentioned Illumination-imaging geometry, multiplereflections are occurring in the walls of the cavity, before the lightreaches the tissue under analysis. In the case of colposcopy, multiplereflections are much more intense, since they are mainly taking placeonto highly reflective blades of the speculum. Recall that the latter isinserted into the vagina to facilitate the inspection of cervix.

[0074] If the illuminator of the imaging apparatus emits linearlypolarized light, the multiple reflections are randomizing thepolarization plane of the incident light. And as it has been discussedabove, if the incident to the tissue under analysis light is notlinearly polarized, then the elimination of the contribution of thesurface reflection to the captured image can not be effective.

[0075]FIG. 7 illustrates an optical imaging apparatus which comprises alight source located at the central part of its front-aperture. Withthis arrangement, the central ray of the emitted light cone is coaxial,with the central ray of the light beam that enters the imagingapparatus. This enables illumination rays to reach directly the tissuesurface under examination and not after multiple reflections in the wallof the cavity. A reflective-objective lens is used, consisted at leastof a first reflection (1RM) and a second reflection (2RM) mirror, whereat the rear part of the first reflection mirror (2RM), a light source(LS) is attached together (if required) with optics for light beammanipulation such as zooming and focusing (SO). The reflective objectivelens (RO), by replacing the common refractive-objective, which is usedin conventional microscopes, provides imaging capability in cavities ofsmall diameter, with freedom in choosing the working distance. Thezooming and focusing optics of the light beam can be adjustedsimultaneously with the mechanism for varying the magnification of theoptical imaging system, so that the illumination area and thefield-of-view of the imaging system, are varying simultaneously andproportionally. This has as a result, the preservation of imagebrightness regardless of the magnification level of the lens. Theimaging-illumination geometry embodied in this optical imaging apparatusamong with the light beam manipulation options, enable the efficientelimination of the contribution of the surface reflection to thecaptured image and consequently the efficient clinical implementation ofthe method described herein.

[0076] Equivalents

[0077] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed:
 1. A method for monitoring the effects of a pathologydifferentiating agent on a tissue sample, comprising: applying apathology differentiating agent on a tissue sample and monitoring therate of change of light reflection from said tissue sample over time,thereby monitoring the effects of a pathology differentiating agent on atissue sample.
 2. The method of claim 1, wherein said pathologydifferentiating agent is acetic acid.
 3. The method of claim 1, whereinsaid tissue sample is a cervical tissue sample.
 4. The method of claim1, wherein said tissue sample is an esophagus tissue sample.
 5. Themethod of claim 1, wherein said tissue sample is an ear tissue sample.6. A method for the in vivo diagnosis of a tissue abnormality in asubject, comprising contacting a tissue in a subject with a pathologydifferentiating agent; exposing said tissue in said subject to opticalradiation; and monitoring the intensity of light emitted from saidtissue over time, thereby diagnosing a tissue abnormality in a subject.7. The method of claim 6, wherein said optical irradiation is broad bandoptical radiation.
 8. The method of claim 6, wherein said opticalirradiation is polarized optical radiation.
 9. The method of claim 6,wherein said tissue abnormality is selected from the group consisting ofa tissue atypia, a tissue dysplasia, a tissue neoplasia and cancer. 10.The method of claim 6, wherein said tissue abnormality is a high gradeneoplasia.
 11. The method of claim 6, wherein said tissue abnormality isa cervical intraepithelial neoplasia.
 12. The method of claim 6, whereinsaid pathology differentiating agent is acetic acid.
 13. The method ofclaim 6, wherein said tissue is a cervical tissue.
 14. The method ofclaim 6, wherein said tissue is an esophagus tissue.
 15. The method ofclaim 6, wherein said tissue is an ear tissue.
 16. The method of claim6, wherein the intensity of light emitted from said tissue over time ismonitored in every spatial point of the tissue.